ProjectSummary Humans, like all mammals, possess limited natural ability to efficiently replace lost myocardium with new contractiletissue.Thisdeficiencycontributestoheartfailure,theleadingcauseofmorbidityandmortalityinthe UnitedStates.Bycontrast,teleostfishefficientlyregeneratenewcardiactissuesafterheartinjury,representing a good model for studying heart repair and regeneration. We are interested to understand how regenerative responsestoinjuryhavebeenoptimizedinadultzebrafish,todiscovernewtargetsthatunderlietheregenerative deficiencies in mammals. In previous work, we have investigated the function of the epicardium on zebrafish heartregenerationandfoundthatepicardialdepletiondecreasesCMproliferation,causesdefectivemyocardial regeneration,andreducescoronaryrevascularization.However,theepicardiumitselfisaheterogeneoustissue and it is not known which epicardial subpopulations offer benefits to heart regeneration. Studies of subpopulations of epicardial cells have been limited by the lack of genetic tools to specifically label and manipulate distinct epicardial cell types. In preliminary studies, we employed deep sequencing, in situ hybridizationandBACtransgenictechnologyinsearchfornovelgeneticmarkersspecificfortheseelusivecell types. We have identified a new transgenic strain hapln1a:EGFP to specifically mark a subpopulation of epicardial cells. We also identified a novel transgenic strain deltaC:EGFP specifically labeling coronary endothelial cells to help define the role of hapln1a+ cells on coronary revascularization. With a panel of new tools, we found that: 1) hapln1a+ cells surround growing cardiomyocytes and could pioneer coronary growth during development;? 2) hapln1a+ cells rapidly accumulate in the sites of cardiac wound prior to myocardial regeneration and coronary revascularization;?and 3) depleting hapln1a+ cells blocks myocardialand coronary growth. In this proposal, we will address central questions about functions of hapln1a+ cells for the two key cardiacregenerativeevents:myocardialregenerationandcoronaryrevascularization.Ouroverallhypothesisis thathapln1a+cellsareaspecializedepicardialsubpopulationcontrollingmyocardialandcoronarygrowthduring development and regeneration. To test this hypothesis, we will: 1)define requirements of hapln1a+ cells for myocardialgrowthduringdevelopmentandregenerationwithnewmethodsofdepletinghapln1a+cells;?2)define requirements of hapln1a+ cells for coronary vascularization and revascularization;? and 3) define molecular nature of hapln1a+ cells during heart regeneration, by utilizing deep sequencing and in situ screen to identify candidategenesandgeneratingknockoutzebrafishwithCRISPR/Cas9technologyforfunctionalanalysis.Our work will generateparadigm-shiftingdiscoveries in theepicardiumand coronary vascular biology. It will reveal the impact of subpopulations of epicardial cells on heart regeneration and key underlying regulators. These findingswillpotentiallyleadtonewtherapeuticstrategieslikeaugmentinghapln1a+cellstoenhancethelimited regenerationdisplayedbyhumansaftermyocardialinfarction.